Methanosarcina mazei Medium (DSMZ Medium 120)

[所属分类:培养基配方] [发布时间:2021-5-26] [发布人:网站管理员2] [阅读次数:] [返回]

Methanosarcina mazei Medium
(DSMZ Medium 120)

山东拓普生物工程有限公司 培养基配方 http://www.topbiol.com

Composition 1112.0mL:

NaCl............................................................................................2.25g
Yeast extract..................................................................................2.0g
Casitone........................................................................................2.0g
NaHCO 3 ......................................................................................0.85g
NH 4 Cl ...........................................................................................0.5g
MgSO 4 ·7H 2 O................................................................................0.5g
K 2 HPO 4 .....................................................................................0.348g
CaCl 2 ·2H 2 O ................................................................................0.25g
KH 2 PO 4 .....................................................................................0.227g
FeSO 4 ·7H 2 O..............................................................................0.002g
Resazurin..................................................................................0.001g
Methanol solution....................................................................20.0mL
L -Cysteine-HCl·H 2 O solution..................................................15.0mL
Na 2 S·9H 2 O solution.................................................................15.0mL
Na-acetate solution..................................................................10.0mL
Vitamin solution.......................................................................10.0mL
NaHCO 3 solution.....................................................................10.0mL
Trace elements solution SL-10..................................................1.0mL
pH 6.5–6.8 at 25°C
Vitamin Solution:
Composition per liter:
Pyridoxamine-HCl...................................................................10.0mg
Pantothenic acid.........................................................................5.0mg
Riboflavin..................................................................................5.0mg
Alpha-lipoic acid .......................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Thiamine-HCl·2H 2 O..................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
Preparation of Vitamin Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Filter sterilize.
Trace Elements Solution SL-10:
Composition per liter:
FeCl 2 ·4H 2 O...................................................................................1.5g
CoCl 2 ·6H 2 O...........................................................................190.0mg
MnCl 2 ·4H 2 O ..........................................................................100.0mg
ZnCl 2 ........................................................................................70.0mg
Na 2 MoO 4 ·2H 2 O.......................................................................36.0mg
NiCl 2 ·6H 2 O..............................................................................24.0mg
H 3 BO 3 ........................................................................................6.0mg
CuCl 2 ·2H 2 O...............................................................................2.0mg
HCl (25% solution)..................................................................10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15
psi pressure–121°C.
NaHCO 3 Solution:
Composition per 100.0mL:
NaHCO 3 ........................................................................................5.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Na 2 S·9H 2 O Solution:
Composition per 100.0mL:
Na 2 S·9H 2 O....................................................................................3.0g
Preparation of Na 2 S·9H 2 O Solution:  Add Na 2 S·9H 2 O to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Before use, neutralize to pH 7.0 with sterile HCl.
Methanol Solution:
Composition per 100.0mL:
Methanol..................................................................................50.0mL
Preparation of Methanol Solution:  Add methanol to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Na-Acetate Solution:
Composition per 100.0mL:
Na-acetate ...................................................................................25.0g
Preparation of Na-Acetate Solution:  Add Na-acetate to distilled/
deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
L -Cysteine Solution:
Composition per 100.0mL:
L -Cysteine·HCl·H 2 O .....................................................................3.0g
Preparation of  L -Cysteine Solution:  Add  L -cysteine·HCl·H 2 O to
distilled/deionized water and bring volume to 100.0mL. Mix thorough-
ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 gas atmosphere. Add components, except vitamin solu-
tion, NaHCO 3 solution, methanol solution,  L -cysteine-HCl·H 2 O solu-
tion, Na 2 S·9H 2 O solution, Na-acetate solution, and trace elements
solution SL-10, to distilled/deionized water and bring volume to 1.0L.
Mix thoroughly. Adjust pH to 6.5. Sparge with 80% N 2 + 20% CO 2 . Au-
toclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobi-
cally add 10.0mL vitamin solution, 10.0mL NaHCO 3 solution, 20.0mL
methanol solution, 15.0mL  L -cysteine-HCl·H 2 O solution, 15.0mL
Na 2 S·9H 2 O solution, 10.0mL Na-acetate solution, and 1.0mL trace el-
ements solution SL-10. Mix thoroughly. Aseptically and anaerobically
distribute into sterile tubes or bottles. After inoculation, flush and re-
pressurize the gas head space of culture bottles with sterile 80% H 2 +
20% CO 2 to 1 bar overpressure.
Use: For the cultivation of Mathanosarcina mazei.